The present invention relates to live attenuated bacteria of the genus Actinobacillus pleuropneumoniae, having a mutation in a gene encoding a toxin, methods for the production of such bacteria, to vaccines comprising such bacteria, methods for the production of such vaccines, to vaccines comprising a toxin, methods for the production of such vaccines and methods for the protection of animals against infection with bacteria of the genus, Actinobacillus pleuropneumoniae. 
Bacteria belonging to the genus Actinobacillus all produce so-called RTX-toxins. (RTX stands for repeat in toxin).
It is the presence of these RTX-toxins that highly contributes to the pathogenic character of these bacteria.
The RTX-toxins have been extensively reviewed by Braun et al. (Critical Rev. in Microbiol. 18(2): 115-158 (1991)). RTX-toxins in Gram-negative strains have also been reviewed in Welch, R. A. (Molecular Microbiology 5/3: 521-528 (1991)) and in Welch et al. (Inf. Agents and Disease 4: 254-272 (1995)).
All known RTX-toxins display some kind of cytotoxic or cytolytic activity. The target-cell-and host-specificity differ however, depending on the toxin and on differences in acylation (McWhinney et al.; J. Bact. 174: 291-297 (1992) and Hackett et al.; J. Biol. Chem. 270: 20250-20253 (1995)). As a result of the difference in target cells, the various toxins of the RTX-toxin family are known e.g. as haemolysin, cytolysin or cytotoxin. The genus Actinobacillus comprises a number of different species, inter alia, Actinobacillus pleuropneumoniae, A. actinomycetemcomitans, A. suis, A. rossii, A. equuli and A. lignieresii. 
Actinobacillus pleuropneumoniae produces serotype-dependent RTX-toxins that are cytotoxic/cytolytic to pig, horse, bovine and human erythrocytes, to rabbit and porcine neutrophils and to porcine alveolar macrophages. (Rosendal et al; Am. J. Vet. Res. 49: 1053-1058 (1988), Maudsley J. R. and Kadis S; Can. J. Microbiol. 32: 801-805 (1986), Frey. J and Nicolet. J; Inf. and Imm. 56:2570-2575 (1988), Bendixon et al; Inf. and Imm. 33: 673-676 (1981), Kamp, E. M. and van Leengoed, L. A. M. G.; J. Clin. Microbiol. 27: 1187-1191 (1989)).
Infections with Actinobacillus in pigs are the cause of severe economic losses to pig industry, due to acute mortality in young pigs and reduced weight gain in older animals.
The genetic organisation of the operons involved in the synthesis, activation and transportation of the RTX toxins in Gram-negative bacteria has been reviewed recently by Coote, J. G. (FEMS Microbiology reviews 88: 137-162 (1992)). Frey has specifically reviewed the known three RTX-toxins in Actinobacillus pleuropneumoniae in Bacterial Protein Toxins, Zbl Bakt. Suppl. 24, p. 322-, Freer et al. (Eds.), Gustaf Fischer, Stutttgart, Jena, New York, 1994.
In Actinobacillus pleuropneumoniae, this kind of RTX-operon contains four genes: the actual Toxin-gene (A), an Activator-gene (C), and two genes (B and D) encoding proteins involved in secretion of the toxin into the surrounding medium. The primary translation-product of the Toxin-gene (A) is a non-toxic protein, of which the toxic activity is activated by the Activator-gene (C) product.
Until recently, it was assumed that only three RTX-toxins, all having the above-described genetic organisation or at least having the Toxin-gene (A) and Activator-gene (C), existed in Actinobacillus species.
These three RTX-toxins, ApxI, Apx-II and Apx-III have respectively a pronounced haemolytic activity (ApxI), a mild haemolytic activity (Apx-II) or a macrophage-cytotoxic activity (Apx-III).
The various toxic activities are fairly randomly divided over the serotypes. There are four subgroups:
a subgroup A, represented by serotypes 1, 5, 9 and 11, producing ApxI and Apx-II,
a subgroup B, represented by serotypes 2, 3, 4, 6 and 8, producing Apx-II and Apx-III,
a subgroup C, represented by serotype 10, producing ApxI only,
a subgroup D, represented by serotype 7 and 12, producing Apx-II only,
It is known, that ApxI, -II, and -III all are essential elements in universal vaccines against Actinobacillus pleuropneumoniae infection: a vaccine not comprising at least ApxI, -II, and -III will not provide protection against all Actinobacillus pleuropneumoniae serotypes. Also, a vaccine not comprising at least the Apx-toxins of one specific serotype will not even induce protection against that single serotype.
Subunit vaccines based on in vitro synthesised RTX-toxins from A. pleuropneumoniae that lost their toxicity have been described earlier, e.g. in European Patent EP No. 0,354,628, in which subunit vaccines based upon a haemolysin and a cytotoxin of A. pleuropneumoniae are disclosed, and in European Patent EP No 0,453,024, in which A. pleuropneumoniae subunit vaccines based upon haemolysins, cytotoxins and outer membrane proteins are disclosed.
There are however four important disadvantages to subunit vaccines in general:
high amounts of antigenic material are needed in order to adequately trigger the immune system.
usually, only B-cell immunity is triggered.
several protective antigens are only triggered in vivo, and therefore can not be present in subunit vaccines.
a live pathogenic bacterium has many important immunogenic molecules, such as Outer Membrane Proteins and capsular polysaccharides, all potentially important for protection and thus to be included in an efficient subunit vaccine.
Next to the obvious problems mentioned under points one and two, especially the fourth point makes it difficult to make an efficient subunit vaccine.
This is e.g. illustrated by the A. pleuropneumoniae subunit vaccine disclosed in European Patent EP No 0,453,024 mentioned above, in which four different subunits (three RTX-toxins and an outer membrane protein) are combined in one vaccine.
It is clear, that in order to overcome the disadvantages of subunit vaccines against Pasteurellacea-infection, a live attenuated vaccine would be highly desirable.
A live attenuated vaccine has the following advantages:
it can be administered in low doses (it is self-replicating)
it closely mimics the natural/wild-type infection
it provides all the possible immunologically important antigens at the same time.
Nevertheless, in spite of the clear advantages, no live vaccines based on Actinobacillus pleuropneumoniae were commercially available prior to the present invention.
The reason for this lies in the following paradox: as mentioned before, ApxI, -II, and -III all are essential elements of universal vaccines against Actinobacillus pleuropneumoniae infection. Live vaccines therefore have to produce these three RTX-toxins. These three RTX-toxins are however strong virulence factors in all Actinobacillus species (see e.g. Coote, J. G.; FEMS Microbiology reviews 88: 137-162 (1992), Tascon et al.; Mol. Microbiol. 14: 207-216 (1994)), Jansen et al.; Inf. and Imm. 63: 27-37 (1995)).
Deletion of the RTX-toxins in order to attenuate the virulence of live App strains is technically feasible, but this does not provide a solution for the dilemma: such RTX-negative strains would be useless as live attenuated vaccine strains since they do no longer induce immunity in the host against the haemolytic/cytotoxic activity of Actinobacillus pleuropneumoniae field strains.
Virulence factors that, although important in the induction of immunity, do play a less important role in building up immunity than ApxI, -II and -III, and thus can in principle be deleted are however currently not known.
It would thus be highly desirable to have a site on the genome of App that attributes to virulence and therefore leads to an attenuated App strain when modified, whereas at the same time it is, although useful in triggering immunity, dispensable from a vaccine point of view. No such sites are however currently known. Moreover, it would be highly desirable if such a site would be universally present in all App strains, instead of being restricted to certain serotypes. Such a site would then allow all different serotypes to be attenuated by deletion of that same site.
It is one of the objectives of the present invention to provide such an attenuation site, universally present in all Actinobacillus pleuropneumoniae strains regardless of their serotype.
Recently, a new gene was found in a serotype 1 strain of Actinobacillus pleuropneumoniae (Thesis T. J. Anderson November 1995).
Although this gene does not resemble the known Actinobacillus ApxI, -II and -III genes, it bears resemblance to RTX-toxin genes known from bacteria belonging to Neisseria meningitidis, for which reason it was named RTX-gene apxlV. The gene however differs in almost all aspects from the three known RTX-toxin genes apxI, -II and -III present in the various species of the Actinobacillus family as described above. First of all, the genomic organisation is completely different. Secondly, there is no activator-mechanism as is found for the known Apx-toxins. In the third place, no specific in vivo haemolytic or cytotoxic activity could at that time be attributed to the gene, or it""s possible gene product.
It was now surprisingly found that this gene, fully in contrast with the three known RTX-genes, is present in all bacteria of the species Actinobacillus pleuropneumoniae, regardless their serotype. This was determined by hybridisation of a probe comprising apxIV coding sequences with restriction fragments of the DNA from Actinobacillus pleuropneumoniae of all serotypes as described in Example 6 and 7.
Unexpectedly it was found now that apxIV deletion mutants are viable, but they behave less virulent compared to their apxIV-possessing parent strains.
Therefore, it was determined that the gene product, the ApxIV toxin is a virulence factor in all Actinobacillus pleuropneumoniae strains. This is an unexpected conclusion, since up until now, no effects at all, let alone effects possibly influencing virulence had been attributed to the gene product in vivo. In fact, up until now there was not even proof that the gene was expressed in Actinobacillus pleuropneumoniae in vivo or in vitro anyway.
It therefore is one of the merits of the invention that it was found that:
the apxIV gene is present in all A. pleuropneumoniae strains regardless the serotype,
the apxIV gene product is a virulence factor in all A. pleuropneumoniae serotypes,
A. pleuropneumoniae strains with a deletion in the apxIV gene are still viable but have a decreased virulence without significantly impairing the immunogenic properties of the strains,
Therefore, the invention provides for the first time live attenuated bacteria of the species Actinobacillus pleuropneumoniae, that do not produce a functional ApxIV toxin.